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Efficient, reproducible Agrobacterium-mediated transformation of sorghum using heat treatment of immature embryos

  • Genetic Transformation and Hybridization
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Abstract

A number of parameters related to Agrobacterium-mediated infection were tested to optimize transformation frequencies of sorghum (Sorghum bicolor L.). A plasmid with a selectable marker, phosphomannose isomerase, and an sgfp reporter gene was used. First, storing immature spikes at 4°C before use decreased frequency of GFP-expressing calli, for example, in sorghum variety P898012 from 22.5% at 0 day to 6.4% at 5 days. Next, heating immature embryos (IEs) at various temperatures for 3 min prior to Agrobacterium infection increased frequencies of GFP-expressing calli, of mannose-selected calli and of transformed calli. The optimal 43°C heat treatment increased transformation frequencies from 2.6% with no heat to 7.6%. Using different heating times at 43°C prior to infection showed 3 min was optimal. Centrifuging IEs with no heat or heating at various temperatures decreased frequencies of all tissue responses; however, both heat and centrifugation increased de-differentiation of tissue. If IEs were cooled at 25°C versus on ice after heating and prior to infection, numbers with GFP-expressing cells increased from 34.2 to 49.1%. The most optimal treatment, 43°C for 3 min, cooling at 25°C and no centrifugation, yielded 49.1% GFP-expressing calli and 8.3% stable transformation frequency. Transformation frequencies greater than 7% were routinely observed using similar treatments over 5 months of testing. This reproducible frequency, calculated as numbers of independent IEs producing regenerable transgenic tissues, confirmed by PCR, western and DNA hybridization analysis, divided by total numbers of IEs infected, is several-fold higher than published frequencies.

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Acknowledgments

S. Gurel (S.G.) thanks The General Directorate of Turkish Sugar Factories Co. for the work permission abroad. E. Gurel (E.G.) deeply appreciates the financial support of TUBITAK (The Scientific and Technological Research Council of Turkey). E.G. and S.G. are also cordially grateful to Professor P.G. Lemaux (P.G.L.) for the opportunity to come to the University of California, Berkeley, as visiting scholars. P.G.L. was supported by USDA Cooperative Extension through the University of California. R. Kaur and J. Wong were supported through a Gates Foundation Grand Challenges for Global Health award to Africa Harvest, Nairobi, Kenya. H.-Q. Tan was supported by the UC Berkeley College of Natural Resources Sponsored Projects for Undergraduate Research. The authors thank Dr. George Liang for the gift of the pGFP-PMI plasmid, Toshihiko Komari and Naoki Takemori from Japan Tobacco Inc., for helpful discussions, Dr. Lixin Zhu at UC Berkeley for the chemiluminescence analysis of GFP and Eric Trieu for technical help with PCR analyses.

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  1. Songul Gurel

    Present address: Department of Plant Breeding, Sugar Institute, Etimesgut, 06790, Ankara, Turkey

  2. Ekrem Gurel

    Present address: Department of Biology, Faculty of Science and Literature, Abant Izzet Baysal University, 5380, Bolu, Turkey

Authors and Affiliations

  1. Department of Plant and Microbial Biology, University of California, 111 Koshland Hall, Berkeley, CA, 94720, USA

    Songul Gurel, Ekrem Gurel, Rajvinder Kaur, Joshua Wong, Ling Meng, Han-Qi Tan & Peggy G. Lemaux

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  1. Songul Gurel
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Correspondence to Peggy G. Lemaux.

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Gurel, S., Gurel, E., Kaur, R. et al. Efficient, reproducible Agrobacterium-mediated transformation of sorghum using heat treatment of immature embryos. Plant Cell Rep 28, 429–444 (2009). https://doi.org/10.1007/s00299-008-0655-1

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